Hydrolysed proteins, which are widely used in the food industry, may be prepared by hydrolysis of protein material with acid, alkali or enzymes. However, on the one hand, acid or alkaline hydrolysis can destroy the essential amino acids produced during hydrolysis thus reducing the nutritional value, whereas enzymatic hydrolysis rarely goes to completion so that the hydrolysed protein contains substantial amounts of peptides.
The filamentous ascomycete Aspergillus oryzae is known to secrete a large variety of amylases, proteinases and peptidases, the action of which are essential for the efficient solubilisation and hydrolysis of raw materials (see WO94/25580). Various methods have been used Aspergillus oryzae for the preparation of food products, especially methods involving the use of a koji culture.
EP417481 (Nestle) thus describes a process for the production of a fermented soya sauce, in which a koji is prepared by mixing an Aspergillus oryzae koji culture with a mixture of cooked soya and roasted wheat, the koji is then hydrolysed in aqueous suspension for 3 to 8 hours at 45.degree. C. to 60.degree. C. with the enzymes produced during fermentation of the Aspergillus oryzae koji culture, a moromi is further prepared by adding sodium chloride to the hydrolysed koji suspension, the moromi is left to ferment and is then pressed and the liquor obtained is pasteurized and clarified.
EP429760 (Nestle) describes a process for the production of a flavouring agent in which an aqueous suspension of a protein-rich material is prepared, the proteins are solubilized by hydrolysis of the suspension with a protease at pH6.0 to 11.0, the suspension is heat-treated at pH 4.6 to 6.5, and the suspension is ripened with enzymes of a koji culture fermented by Aspergillus oryzae.
Likewise, EP96201923.8 (Nestle) describes a process for the production of a meat flavour, in which a mixture containing a vegetal proteinaceous source and a vegetale carbohydrates containing source is prepared, said mixture having initially at least 45% dry matter, the mixture is inoculated with a koji culture fermented by Aspergillus oryzae and by one or more another species of microorganisms involved in the traditional fermentation of meat, and the mixture is incubated until meat flavours are formed.
Depending on the nature of the protein and the enzymes used for proteolysis, the peptides formed can however have extremely bitter tastes and are thus organoleptically undesirable. There is hence a need for methods of hydrolysing proteins leading to high degree of protein hydrolysis and to hydrolysates with excellent organoleptic properties.
In addition, in protein rich materials subjected to enzymatic hydrolysis, a high level of glutaminase is required to convert glutamine into glutamic acid which is an important natural taste enhancer (see WO95/31114). Biochemical analysis of residual peptides in cereals hydrolysed by Aspergillus oryzae, i.e. wheat gluten, shows however that a considerable amount of glutamine remains sequestered in proline containing peptides (Adler-Nissen, In: Enzymatic hydrolysis of food proteins. Elsevier Applied Sciences Publishers LTD, p120, 1986). There is hence a need for methods of hydrolysing proteins leading to liberation of high amount of glutamine.
Among the different proteases known from koji molds, two neutral endopeptidase (Nakadai et al., Agric. Biol. Chem., 37, 2695-2708, 1973), an alkaline endopeptidase (Nakadai et al., Agric. Biol. Chem., 37, 2685-2694, 1973), an aspartic protease (Tsujita et al., Biochem. Biophys Acta, 445, 194-204, 1976), several aminopeptidases (Ozawa et al., Agric. Biol. Chem., 37, 1285-1293, 1973), several carboxypeptidases (Nakadai et al., Agric. Biol. Chem., 37, 1237-1251, 1970) have been identified and purified.
More recently a prolyl-dipeptidyl-peptidase activity has been detected in Aspergillus oryzae, which is an enzyme providing a high level of hydrolysing specificity towards proteins and peptides starting with X-Pro- thus liberating dipeptides of X-Pro type, wherein X is any amino-acid (Tachi et al., Phytochemistry, 31, 3707-3709, 1992).